1. Field of the Invention
The present invention relates to microscopic imaging, and, more particularly, to holographic microscopy for reconstruction of three-dimensional objects and optical tomographic imaging for selectively imaging cross sections of an object.
2. Description of Related Art
Imaging of microscopic objects is an essential art, not only in biology and medicine, but also in many other fields of science and technology, including materials science, microelectronics engineering, and geology. Modern microscopy takes advantage of discoveries on the interaction between electromagnetic and other fields with material objects, and has taken on numerous incarnations, such as electron transmission and scanning microscopes, the scanning tunneling microscope, the atomic force microscope, and the laser scanning confocal microscope (Isenberg, 1998).
Rapid progress in electronic detection and control, digital imaging, image processing, and numerical computation has been crucial in advancing modern microscopy. By equipping an optical microscope with a digital or video camera, a range of image processing and pattern recognition techniques can be applied for automated image acquisition and analysis (Herman and Lemasters, 1993).
One particular aspect of microscopic imaging of interest is the axial resolution or depth of focus. In a conventional optical microscope, the lateral resolution can be a fraction of a micrometer, whereas the axial resolution is typically several micrometers or more. This leads to two related difficulties: One is that the axial position cannot be determined with better than a few-micrometer accuracy; another is that the overlap of images from object planes several micrometers apart leads to blurring and degradation of images. Usually the only remedy available is physical sectioning of the specimen into thin slices, which precludes a large range of materials from being studied.
A remarkable solution to these problems was the scanning confocal microscope, developed just over a decade ago (Sheppard and Shotton, 1997). by illumination of a single object point and placement of a detection aperture at the image point, the detector behind the aperture registers a light signal originating only from the object point. A two- or three-dimensional image is constructed by pixel-by-pixel scanning of the object volume. The whole of the resulting image is sharply in focus, and the size of the acquirable image is limited only by the stability and speed of the scanning and processing system.
Another important optical scanning system is the near-field optical scanning microscope, where the light signal is probed by a highly tapered optical fiber at a distance only a fraction of an optical wavelength from the sample surface, thereby circumventing the diffraction limit of resolution in far-field imaging. However, the application of this technique in imaging wet and delicate biological samples has been limited because of the requirement to maintain a constant surface-probe distance with accuracy and stability. It is more suitable for the study of macromolecular structures, as with other related scanning devices that utilize electron tunneling, atomic force, and other subtle interactions on the atomic and molecular level.
Holography was originally invented in an attempt to improve the resolution of a microscope (Hariharan, 1996). Both the amplitude and phase information of the light wave are recorded in a hologram by the interference of an object wave that is to be imaged with a reference wave of simple structure such as a plane or spherical wave. The interference pattern is recorded in a variety of media, most commonly on a photographic plate. The object wave is reconstructed as one of the diffraction patterns when a replica of the reference wave is incident on the photographic plate. The resulting image is an exact copy of the light wave that originally emanated from the object, and thus has the property of perspective vision.
Because the holographic image retains the phase as well as the amplitude information, a variety of interference experiments can be performed, and this is the basis of many interferometric applications in metrology. It is possible accurately to measure deformation and other variations of an object at a submicrometer level because of advances in digital imaging and numerical computing technology. Thus it is often advantageous to replace steps of the holographic procedure with digital processes (Yaraslavskii and Merzlyakov, 1980).
In computer-generated holograms (CGH) the interference pattern is computed from a mathematical definition of a virtual object and reference (Trester, 1996). The patter is output to a hard-copy device, and laser illumination results in an optical hologram image.
On the other hand, in computer-reconstructed holograms (CRH), the optical interference pattern of a real object and reference is recorded using an electronic or digital camera (Schnars and Jüptner, 1996). The pattern is digitized and stored in a computer, and the holographic image is recreated on the computer by numerical calculation.
In either CGH or CRH, the numerical calculation basically imitates the optical diffraction process as the light wave propagates from the object to the hologram plane or from the hologram plane to the image plane. This can be accomplished using Fresnel diffraction theory or Huygens wavelet theory (Kreis et al, 1997). An important aspect of research in this area is in attempts to minimize the computational load using, for example, segmentation of holograms and horizontal-only parallax (Karnaukhov et al., 1998; Yang et al., 1998a,b).
Digital holography alleviates the need for wet chemical processing of a photographic plate, although at some expense of resolution. However, once the amplitude and phase (i.e., all the essential information) of the light wave are recorded numerically, one can easily subject these data to a variety of manipulations, and so digital holography offers capabilities not available in conventional holography. For example, the phase information of the light wave is available directly from the numerical reconstruction and greatly simplifies interferometric deformation analysis (Seebacher et al., 1998; Kreis et al, 1998; Cuche et al, 1999; Brown and Pryputniewicz, 1998).
Holography can be applied to microscopy in two alternative ways. In one, a hologram of a microscopic object is taken directly, and the hologram is inspected using a microscope; in the other, a microscope is used first to magnify the object image, and the hologram is taken of that image. Holographic microscopy has been particularly useful in particle analysis, where a particle count has to be obtained in a volume of fluid (Vikram, 1992). With a conventional microscope, the constant motion of particles into and out of the focal plane makes it difficult to ascertain an accurate count as the focal plane is scanned across the entire sample volume. A holographic micrograph freezes the three-dimensional field, and a particle count can proceed by focusing on successive planes.
Holographic microscopy in three-dimensional imaging applications has been limited partly because of the inherent scale distortion of an optical microscope image of a volume object. The axial magnification goes as the square of the lateral magnification, so that the two directions magnify with different ratios, and the lateral magnification also depends on the axial distance. When the hologram is viewed by focusing on a plane, the same problem of out-of-focus image blurring is present as in an optical microscope (Zhang and Yamaguchi, 1998; Poon et al., 1995).
Application of digital holography in microscopy holds potentially attractive benefits (Schilling et al., 1997). In principle, once the amplitude and phase information of the object image is numerically stored, it can be manipulated by image processing techniques for removal of distortion and out-of-focus blurring. Interference measurements can yield subwavelength resolution of features, and particle analysis and feature recognition can be automated with greater efficiency.
Another imaging technique, tomography, has been utilized in biomedical and materials sciences (Robb, 1997), with optical tomography most useful in microscopic imaging because of the short wavelength and limited penetration depth of most biological surfaces. For example, laser confocal microscopy (Sheppard and Shotton, 1997) uses aperturing of both the illuminated sample volume and the detector aperture, thereby rejecting all scattered light other than from the focal volume. Optical coherence tomography (Huang et al., 1991; Morgner et al., 2000) is a time-of-flight measurement technique, using ultrashort laser pulses or a continuous-wave laser of very short coherence time. In both of these methods the signal is detected one pixel at a time, and the three-dimensional image is reconstructed by scanning the three-dimensions pixel by pixel. Although microscanning using piezo actuators is an important technique, being able to obtain image frames at a time would have technical advantages.
By recording the phase as well as the intensity of light waves, holography allows reconstruction of the image of 3D objects, and gives rise to many metrological and optical processing techniques (Hariharan, 1996). It is now possible to replace portions of the holographic procedure with electronic processes (Yaroslavsky and Eden, 1996). For example, in digital holography the hologram is imaged on a CCD array, replacing photographic plates of conventional holography. The digitally converted hologram is stored in a computer, and its diffraction is numerically calculated to generate simulation of optical images.
With digital holography, real-time processing of the image is possible, and the phase information of the reconstructed field is readily available in numerical form, greatly simplifying metrological applications (Cuche et al., 1998). Previously limiting memory and speed factors have improved (Trester, 1996; Piestun et al., 1997). On the other hand, for the purpose of tomographic imaging, although the hologram produces a 3D image of the optical field, this does not by itself yield the tomographic distance information to the object surface points, other than by focusing and defocusing of the object points, which is really a subjective decision (Poon et al., 1995; Zhang and Yamaguchi, 1998a). The distance information can be obtained in time-of-flight-type measurements, or it can be determined by counting the number of wavelengths or some multiples of it, which is the basis of various interference techniques.
One technique is the interference of two holograms recorded at two different wavelengths, resulting in a contour interferogram with the axial distance between the contour planes inversely proportional to the differences in wavelengths. In digital holography, it is possible to extend the process to recording and reconstruction of many holograms without introducing any wavelength mismatch or crosstalk. If a number of regularly spaced wavelengths are used for recording and reconstruction, then the peaks of the cosine-squared intensity variation of two-wavelength interference become sharper and narrower, as when a number of cosines with regularly spaced frequencies are added.